Sequence Heterogeneity in Closed Simian Virus 40 Deoxyribonucleic Acid
Sequence Heterogeneity in Closed Simian Virus 40 Deoxyribonucleic Acid
复制标题
闭合猿猴病毒 40 脱氧核糖核酸的序列异质性
作者：
H. Tai;C. Smith;P. Sharp;J. Vinograd
通过甲酰胺-蛋白胶片电子显微镜检查由封闭病毒 DNA 制备的变性、单切口猿病毒 40 (SV40) 脱氧核糖核酸 (DNA) 自退火形成的异源双链分子，以测试 DNA 的序列同质性。序列不均匀性在异源双链体中表现为单链环。如果涉及超过 50 个核苷酸的区域，则这些是由序列删除或序列替换引起的。来自病毒的未变性 DNA 在感染复数下传代两次，每个细胞远小于 1 噬菌斑形成单位 (PFU)，其大小似乎是均匀的。由该 DNA 形成的异源双链体表明大约 2% 的分子带有缺失，但替换低于检测水平。相比之下，通过传代未稀释的裂解物七次或通过以每个细胞5 PFU的感染复数感染原种病毒而生长的病毒的未变性DNA包含大量短于全长的分子。异源双链体样品表明，12% 和 7% 的未变性材料含有碱基取代，13% 和 11% 含有缺失。缺失和取代似乎发生在单独的分子中。对显示取代环特征的异源双链体的长度测量已确定这些分子来自真实的序列取代，而不是来自相邻或重叠的缺失。超过 80% 的带有取代基的分子比天然 SV40 长度短。平均而言，取代序列约为 SV40 长度的 20%，但它取代的序列约为天然长度的 30%。取代的序列可以是宿主细胞核DNA，可能是由于SV40整合到染色体中，然后将SV40 DNA与染色体DNA一起切除而产生的。
The heteroduplex molecules formed by self-annealing of denatured, singly nicked simian virus 40 (SV40) deoxyribonucleic acid (DNA) prepared from closed viral DNA were examined by formamide-protein film electron microscopy to test the DNA for sequence homogeneity. Sequence inhomogeneity appears in the heteroduplexes as single-strand loops. These result from sequence deletion or from sequence substitution, if regions greater than 50 nucleotides are involved. The undenatured DNA from viruses passaged twice at multiplicities of infection much less than 1 plaque-forming unit (PFU) per cell appeared to be homogeneous in size. The heteroduplexes formed by this DNA indicated that approximately 2% of the molecules carried deletions, but that substitutions were below the level of detection. In contrast, undenatured DNA from viruses grown by passaging undiluted lysates seven times or by infection with stock virus at a multiplicity of infection of 5 PFU per cell contained a large frequency of molecules shorter than the full length. The heteroduplex samples indicated that 12 and 7% of the undenatured material contained base substitutions, and 13 and 11% contained deletions. The deletions and substitutions appear to occur in separate molecules. Length measurements on heteroduplexes displaying the loop characteristic of substitutions have established that these molecules are from true sequence substitutions, and not from adjacent or overlapping deletions. More than 80% of the molecules carrying substitutions are shorter than the native SV40 length. On the average, the substituted sequence is about 20% of the length of SV40, but it replaces a sequence about 30% of the native length. The substituted sequences may be host cell nuclear DNA, possibly arising from integration of SV40 into the chromosome followed by excision of the SV40 DNA together with chromosomal DNA.